CEA-binding protein and uses thereof

ABSTRACT

Disclosed is a protein that specifically binds carcinoembryonic actigen (CEA) in the presence of divalent cation in vitro. This protein has a molecular weight of about 20 kD as determined by SDS-polyacrylamide gel electrophoresis, is glycosylated, and includes the amino acid sequence set forth in the Sequence Listing as SEQ ID NO:4. Also disclosed are antibodies that recognize the CEA binding protein, methods of detecting carcinoma, methods of treating carcinoma, and a kit for screening a patient for carcinoma.

REFERENCE TO RELATED APPLICATIONS

This application is related to applicants' copending U.S. patentapplications Ser. No. 708,885, entitled "Method for IsolatingCEA-Binding Protein" and U.S. Ser. No. 714,386, entitled "BindingProtein for CEA and Uses Thereof", both filed on even date herewith.

BACKGROUND OF THE INVENTION

The present invention relates to the field of cancer diagnosis andtreatment, and more particularly to methods of detecting and treatingcarcinoma that elicit carcinoembryonic antigen (CEA). In particular,this invention relates to the identification of new cancer markers, CEAbinding proteins, and methods of isolating such CEA-binding proteins,antibodies specific for these proteins, as well as methods useful in thediagnosis, detection, and monitoring of carcinoma.

Colorectal carcinoma is a cancer which affects approximately 600,000additional people worldwide per year. The prognosis is poor in about 50%of the cases because the tumor is often not detected until the diseasehas spread and has reached a terminal stage. Early diagnosis isimportant to increase chances of arresting the carcinoma before itmetastasizes, thereby leading to an improved prognosis.

A widely used method of the identification of cancerous tissue is todetermine its structural resemblance to fetal or immature tissue. Inthis way, tumors can be classified depending on the degree of cellulardifferentiation; they can be undifferentiated, poorly differentiated,moderately differentiated or well differentiated. In addition, thebehavior of a given tumor can often be related to its degree ofdifferentiation. For example, poorly differentiated tumors tend to growmore rapidly and metastasize earlier than do differentiated tumors whichmore closely resemble the tissue of origin. Poorly differentiated tumorstend to have a poor prognosis and are difficult to detect.

One method of early tumor diagnosis is detection of the Presence of amarker or antigen specific for a particular tumor. These normallyproteinaceous markers are synthesized by the tumor, and may be found inthe serum and/or on the surface of the tumor. Only a limited number oftumor markers for colorectal carcinoma have thus far been found to haveclinical use. These include CEA and the sialyated Lewis a antigen (CA19.9). Unfortunately, approximately 40% of patients whose condition hasbeen accurately diagnosed as colorectal carcinoma do not have elevatedplasma levels of either of these antigens when initially examined. Inthe case of CEA, this may be because this antigen is so rapidly clearedfrom the circulation. Recently, however, two new cancer markers, thecarcinoma orosomucoid-related antigen (CORA) (U.S. Pat. No. 4,914,021)and the CC-glycoprotein (U.S. Pat. No. 4,921,789) have been discoveredby applicants. However, there is no commercially availableserodiagnostic marker which can be used to detect the tumor and tomonitor therapy for this group.

Production of some tumor markers e.g., CEA and CA 19.9, by tumor cellsin vitro correlates with a greater degree of cellular differentiation.For example, CEA and CA 19.9 are present to a far lesser degree onpoorly differentiated or undifferentiated cancer cells than on thosewhich are more differentiated. Accordingly, many patients withundifferentiated colorectal carcinomas never develop elevated serumlevels of either of these markers, even in the terminal stages of thecancer. There is also considerable overlap between the presence of CA19.9 and CEA, the patient with a normal CEA level and an elevated levelof CA 19.9 being the exception rather than the rule. Therefore, newmarkers suitable for identifying and monitoring undifferentiated tumorswould be of great value.

Accordingly, it is an object of this invention to provide new markersfor the detection of carcinoma.

It is another object of the invention to provide new markers suitablefor diagnosing and monitoring, and treatment of carcinoma.

Yet another object is to provide antibodies specific for these newmarkers.

Still another object is to provide a method of isolating CEA-bindingproteins that eliminates contamination by CEA.

A further object of the present invention is to provide a method and kitfor the detection and monitoring of carcinoma in patients usingantibodies specific for markers on carcinoma cells.

Yet another object of the invention is to provide a hybridoma whichproduces a monoclonal antibody that recognizes both undifferentiated andpoorly differentiated carcinoma cells.

A still further object is to provide screening procedures for detectingthe presence of carcinoma cells at all stages of differentiation.

These and other objects of the invention will be apparent from thedescription, drawing and claims which follow.

SUMMARY OF THE INVENTION

New tumor markers for carcinoma have been discovered which have theability to bind CEA. These markers include two CEA-binding proteins(CBPs) having molecular weights of about 20,000 daltons (20 kD) andabout 21 kD as determined by sodium dodecyl sulfate-polyacrylamideelectrophoresis (SDS-PAGE), and binding CEA in vitro in the presence ofa divalent cation. The 20 kD protein includes the amino acid sequenceset forth in the Sequence Listing as SEQ ID NO:4. The 21 kD protein isglycosylated and includes the amino acid sequence set forth in theSequence Listing as SEQ ID NO:2.

This invention also encompasses CEA-binding fragments of these tumormarkers. The term "CEA-binding protein" or "CBP" is used herein todescribe both proteins and fragments thereof which bind CEA.

In one embodiment of the invention, the 20 kD or 21 kD CBP furtherincludes a label, such as one selected from the group consisting ofradioactive isotopes, enzymes, stable free radicals, coenzymes,fluorescent groups, chemiluminescent groups, toxins, and colorimetricgroups. In another embodiment, the 20 kD or 21 kD CBP is bound to asupport which forms a device useful, for example, in purifying CEA.

In yet another embodiment, antibodies and binding fragments thereofspecific for a CBP are provided. Preferably, the antibody is amonoclonal antibody. The antibody can form part of a kit for screening apatient for carcinoma. This kit further includes an antibody specificfor a carcinoma marker selected from the group consisting of carcinomaorosomucoid-related antigen (CORA), the CC glycoprotein,carcinoembryonic antigen (CEA), CA 19.9, non-specific cross-reactingantigen (NCA), and alpha 1-acid glycoprotein (AGP), serum amyloid Pprotein (SAP), and C-reactive protein (CRP).

The invention also provides methods of isolating and using CBPs. Morespecifically, a method of isolating a CBP, such as the 20 kD, the 21 kD,or any CBP that binds CEA in vitro in the presence of a divalent cation,is provided which includes the following steps. A biological samplecontaining CEA and a CBP is provided. The biological sample may beascites fluid, whole blood, serum, bile, saliva, sputum, lymphoidtissue, or tumor tissue obtained from a subject afflicted withcarcinoma. This sample is contacted with a divalent cation, such as Ca⁺2, Zn⁺ 2, or Mg⁺ 2, at a concentration and for a time sufficient toenable the CBP to bind to CEA, thereby forming a CBP-CEA conjugate. Thesample is contacted with an adsorbent that binds CEA, such as animmobilized immunoadsorbent including an antibody to CEA, for a timesufficient to allow the conjugate to adsorb to the adsorbent. Portionsof the sample which have not adsorbed to the adsorbent are removed. TheCBP is then dissociated from the adsorbent-bound conjugate. Dissociationcan be accomplished with the use of an agent that chelates divalentcations such as ethylenediaminetetraacetic acid (EDTA) or ethyleneglycol-bis[β-aminoethyl ether]-N,N,N',N'-tetraacetic acid (EGTA). Thedissociated CBP is then collected, for example, by executing apurification method such as high pressure liquid chromatography (HPLC),SDS-PAGE, affinity chromatography, exclusion chromatography, ammoniumsulfate precipitation, ultracentrifugation, and/or isoelectric focusing.For samples which do not contain CEA, CBP can be isolated by added CEAto the sample, or by reading the sample with CEA immobilized on a solidsupport in the presence of a divalent cation.

A method of detecting carcinoma is provided which includes the followingsteps. A pharmaceutical formulation including the 20 kD or 21 kD CBP, orCEA-binding fragments thereof, is administered in a physiologicallyacceptable carrier to the subject. A biological sample is taken and theconcentration of CEA assayed for CEA. The concentration of CEA is thencompared with a predetermined threshold level of CEA indicative of thepresence of carcinoma.

Another aspect of the invention includes a method of screening forcarcinoma including subjecting a biological sample from a subject to atest, such as an immunoassay, indicating the presence of a cancermarker, and screening the sample for the presence of a CBP, its presencebeing indicative of the presence of carcinoma.

Other embodiments of the invention include assay formats detecting CEAin a body fluid by adsorbing CBP to a support (such as a microtiterplate, for example) treating the body fluid to be screen with divalentcation, adding the treated body fluid to the CBP-bound plate, and thenquantitating the bound CEA with labelled anti-CEA antibody.Alternatively, anti-CEA antibody can be adsorbed to the support which isthen treated with the body sample in the presence of divalent cation.The presence of complexed CEA (or CBP to which the CEA in the bodysample is complexed in the presence of divalent cation) can then bedetected by treatment with labelled anti-CBP antibody. CBPs free in bodysamples can be detected by adsorbing CEA to a support, and then treatingthe support with the body sample in the presence of divalent cation.

Also provided is a method of treating carcinoma. This method includesproviding a CBP and administering it in a pharmaceutically acceptablecarrier to a subject afflicted with carcinoma. The CBP binds CEA presentin the subject, thereby inhibiting the metastatic proliferation ofcarcinoma cells which occurs as a result of CEA binding to CEA receptorson various organs. The provision of the CBP can be accomplished byrecombinant DNA technology, automated or manual biochemical peptidesynthesis, or by purification from a subject inflicted with carcinoma.

BRIEF DESCRIPTION OF THE DRAWING

The foregoing and other objects of this invention, the various featuresthereof, as well as the invention itself may be more fully understoodfrom the following description when read together with the accompanyingdrawings in which:

FIG. 1 is a diagrammatic representation of the CBP purification schemeof the invention;

FIG. 2 is a photographic representation of a stained transfer blot of anSDS gel showing the 20 kD and 21 kD CBPs (lane 3), alpha 1-acidglycoprotein (lanes 1 and 4), column wash (lane 2), and human serumalbumin (lane 5);

FIG. 3A-D is an optical scan of an HPLC of (A) CEA, (B) CBP 41 kDcomplex, (C) CEA+CBP in the presence of CaCl₂, and (D) CEA+CBP in thepresence of EDTA;

FIG. 4A-B is an optical scan of an HPLC of (A) alpha 1-acid glycoprotein(AGP)+CEA in the presence of EDTA, and (B) CEA+CBP in the presence ofCaCl₂ ;

FIG. 5 is a comparison of the amino acid sequences of C-reactive proteinand the 20 kD and 21 kD CBPs; and

FIG. 6 is a comparison of the amino acid sequences of serum amyloid Pprotein and the 20 kD and 21 kD CBPs.

DESCRIPTION OF THE INVENTION

Proteins that influence the concentration of CEA in the blood have beenisolated from patients afflicted with carcinoma. These CEA-bindingproteins are also markers for carcinoma, and as such are useful in thedetection and treatment of carcinoma.

The CBPs have been isolated using a purification method which virtuallyeliminates contamination by CEA. This procedure is schematicallyrepresented in FIG. 1. A biological sample containing CEA and CBP isprovided. The biological sample may be ascites fluid, whole blood,serum, bile, saliva, sputum, lymphoid tissue, or tumor tissue obtainedfrom a subject afflicted with carcinoma. This sample is contacted with adivalent cation, such as Ca⁺ 2, Zn⁺ 2, or Mg⁺ 2, at a concentration andfor a time sufficient to enable the CBP to bind to CEA, thereby forminga CBP-CEA conjugate. The sample is contacted with an adsorbent thatbinds CEA, such as an immobilized immunoadsorbent including an antibodyto CEA, for a time sufficient to allow the conjugate to adsorb to theadsorbent. Portions of the sample which have not adsorbed to theadsorbent are removed. The CBP is then dissociated from theadsorbent-bound conjugate. Dissociation can be accomplished with the useof an agent that chelates divalent cations such asethylenediaminetetraacetic acid (EDTA) or ethyleneglycol-bis[β-aminoethyl ether]-N,N,N',N'-tetraacetic acid (EGTA). Thedissociated CBP is then collected, for example, by executing apurification method such as high performance liquid chromatography(HPLC), SDS-PAGE, affinity chromatography, exclusion chromatography,ammonium sulfate precipitation, ultracentrifugation, and/or isoelectricfocusing.

The instant purification procedure does not depend on the size of thebinding protein, a particularly important point since CBPs isolated bythis method have a molecular weight (by HPLC) similar to that of alpha-1acid gylcoprotein (AGP). The procedure is ligand-specific, and thus anyproteins that may bind to CEA nonspecifically or which require someother binding factors will not contaminate the product. Any contaminatesare removed during the washing or will remain bound to CEA after theEDTA elution. This method also provides an effective way to isolate CBPsfrom CEA to which they have bound. Because CEA is a large molecule andvery immunogenic, it is important to remove it from the CBP isolate ifantibodies to these CBPs are to be prepared from the isolate.

Using this method, two CBPs were isolated, one having a molecular weightof 20 kD and one glycoprotein having a molecular weight of 21 kD, asshown in FIG. 2, lane 3, of a stained transfer blot of an SDS-PA gel.These molecular weights differ from those of other known CEA bindingproteins such as the 46-50 kD carcinoma orosomucoid-related antigen(CORA) (described in U.S. Pat. No. 4,914,021), and also differ from the41 kD alpha 1-acid glycoprotein (AGP) which does not bind CEA.

The physical properties of the 20 kD and 21 kD CBPs, as well as thedegree of purity after isolation, were determined by high pressureliquid chromatography (HPLC). FIGS. 3A-3D show that the proteinsisolated have a collective molecular weight of 41 kD (FIG. 3B). Theappearance of a single peak with a retention time of about 8.5 min andthe absence of a peak at about 9.4 min suggests the formation of acomplex between CEA and CBP in the presence of calcium ions (FIG. 3C).However, in the presence of a calcium chelator like EDTA for example,two peaks with retention times similar to that of CEA (FIG. 3A) and thatof the uncomplexed CBPs (FIG. 3B) were observed (FIG. 3D), indicatingthat these CBPs do not bind CEA in the absence of calcium.

These isolated CBPs were further distinguished from AGP by HPLC. Likethe CBPs, AGP does not bind CEA in the absence of calcium (presence ofEDTA) (FIG. 4A). However, unlike the CBPs, AGP does not bind CEA in thepresence of calcium ions (FIG. 4B). These CBPs have also beendistinguished from CRP and SAP by isoelectric focusing.

A partial sequence of the 20 kD and 21 kD CBPs was obtained and is setforth in the Sequence Listing as SEQ ID NO:2 and SEQ ID NO:4,respectively. These partial sequences were found to have no homologywith CEA or AGP. However, some sequence homology was found with theserum amyloid P protein (SAP) (FIG. 5), and with the C-reactive protein(CRP) (FIG. 6). These sequence analyses were performed using theWisconsin Program Protein Data Base (Genetics Computer Group, Universityof Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis.53705).

SAP is a normal plasma component. It is an α-1 glycoprotein composed often 23-25.5 kD subunits non-covalently linked as a double pentamer. Theserum level of SAP in patients with various clinical types ofamyloidosis, connective tissue diseases, and bacterial pneumonia doesnot differ significantly from normal values. Likewise, the serum levelof patients with carcinoma of the colon do not differ from healthyindividuals in the serum level of SAP (Levo et al. (1982) J. Immunol.Meth. 50:17-31). However, patients with carcinoma of the breast do havesignificantly increased serum concentrations of SAP which correlate withthe severity of the disease (Levo et al. (Scand. J. Immunol. (1986)24:147-151).

CRP, a dipentamer of 21 kD subunits, has a strong sequence homolgy withSAP. Both proteins undergo calcium-dependent ligand binding, arecomposed of non-covalently linked subunits, and share a similarpentagonal disc-like molecular form (Pepys (1982) Eur. J. Rheumatol.Inflamm. 5:386). In addition, they share at least about 60% homology ofamino acid sequence. However, unlike SAP, CRP is an acute phase reactantin humans. Its concentration rises rapidly following acute tissueinjury, infection, or inflammation, and it is often persistentlyelevated in cases of malignant neoplasia.

The sequence homology between the 20 kD and 21 kD CBPs, and CRP and SAP,respectively, raises the possibility that these proteins are related instructure and/or function as cancer markers.

Among the uses of the 20 kD and 21 kD CBPs are methods of detecting andtreating carcinoma. The method of detection involves the administrationto the subject of a pharmaceutical formulation including the 20 kD or 21kD CBP, or CEA-binding fragments thereof, in a physiologicallyacceptable carrier. A biological sample is then taken. This sample maybe ascites fluid, whole blood, serum, bile, saliva, sputum, lymphoidtissue, or tumor tissue. The concentration of CEA in this sample ismeasured. This measurement can be accomplished by any number of knowntests for CEA including an immunoassay (e.g., Roche ELISA, Hoffman-LaRoche, Nutley, N.J.) activity assay, immunoassay, quantitativeelectrophoresis, and the like. The concentration of CEA in thebiological sample is then compared with a predetermined threshold levelof CEA indicative of carcinoma. This threshold CEA concentration can bedetermined by administering a CBP to two statistically significantgroups of people, one with carcinoma, and one that is disease-free. Byway of example, the value of the threshold level may be the point atwhich the measured CEA concentration curves of these two groupsintersect.

CBPs may also be used in the treatment of carcinoma as follows. A CBP isadministered in a pharmaceutically acceptable carrier to a subjectafflicted with carcinoma. The CBP binds CEA present in the subject,thereby inhibiting the metastatic proliferation of carcinoma cells whichoccurs as a result of CEA binding to CEA receptors on various organs(see, e.g., Toth et al. (1985) Cancer Res. 43:342-397; Toth et al.(1988) Biochem. Soc. Trans. 16:1027-1028; Toth et al. (1989) J.Leukocyte Biol. 45:370-376; and Hostetter et al. (1990) J. Natl. CancerInsti. 82:380-385). The concentration of calcium in various body tissuesis high enough to enable efficient binding.

The provision of a CBP in both methods of use can be accomplished bypurification from a subject inflicted with carcinoma. Alternatively,given the sequence of these proteins isolated from body tissues, theCBPs may be prepared by recombinant DNA technology (see. e.g., Maniatiset al., Molecular Cloning, A Laboratory Manual (1982) Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.), or by automated or manualbiochemical peptide synthesis.

Effective dosages of the CBPs and modes of their administration in thedetection and treatment of carcinoma can be determined by routineexperimentation. The pharmaceutical formulation suitable for injectableuse include sterile aqueous solutions or dispersions and sterile powdersfor the extemporaneous preparation of sterile injectable solutions ordispersions. In all cases the formulation must be sterile and must befluid to the extent that easy syringability exists. It must be stableunder the conditions of manufacture and storage and may be preservedagainst the contaminating action of microorganisms, such as bacteria andfungi. The carrier can be a solvent or dispersion medium containing, forexample, water, polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycol, and the like), suitable mixtures thereof, andvegetable oils. The proper fluidity can be maintained, for example, bythe use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In some cases, it may be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable CBPs can be brought about by the use in the compositionsof agents delaying absorption.

Sterile injectable solutions are prepared by incorporating the CBPs inthe required amount in the appropriate solvent with various of the otheringredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thevarious sterilized active ingredients into a sterile vehicle whichcontains the basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum-drying and freeze-drying techniques which yield apowder of the active ingredient plus any additional desired ingredientfrom previously sterile-filtered solution thereof.

The CBP may be administered parenterally or intraperitoneally. Solutionsof the CBP as pharmacologically acceptable salts can be prepared inwater suitably mixed with a surfactant, such as hydroxypropylcellulose.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, and mixtures thereof and in oils. Under ordinary conditions ofstorage and use, these preparations contain a preservative to preventthe growth of microorganisms.

The CBP also may be orally administered, for example, with an inertdiluent or with an assimilable edible carrier, or enclosed in hard orsoft shell gelatin capsules, or they may be compressed into tablets, orincorporated directly with the food of the diet. For oraladministration, the CBP may be incorporated with excipients and used inthe form of ingestible tablets, buccal tablets, troches, capsules,elixirs, suspension syrups, wafers, and the like. Such compositions andpreparations should contain at least 0.1% of the CBP. The percentage ofthe compositions and preparations may, of course, be varied. The amountof CBP in such useful compositions is such that a suitable dosage willbe obtained.

The tablets, troches, pills, capsules and the like may also contain thefollowing: excipients, such as dicalcium phosphate; a disintegratingagent; and a sweetening agent, such as sucrose, lactose or saccharin maybe added or a flavoring agent, such as peppermint, oil of wintergreen,or cherry flavoring. When the dosage unit form is a capsule, it maycontain, in addition to materials of the above type, a liquid carrier.Various other materials may be present as coatings or to otherwisemodify the physical form of the dosage unit. For instance, tablets,pills, or capsules may be coated with shellac, sugar, or both. A syrupor elixir may contain the active compounds sucrose as a sweeteningagent, methyl and propylparabens as preservatives, a dye and flavoring,such as cherry or orange flavor. Of course, any material used inpreparing any dosage unit form should be pharmaceutically pure andSubstantially non-toxic in the amounts employed. In addition, the CBPmay be incorporated into sustained-release preparations andformulations.

As used herein, "pharmaceutically acceptable carrier" includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

Antibodies to the 20 kD and 21 kD proteins are also provided by theinvention. Such antibodies can be easily produced by one with ordinaryskill in the art. For example, a purified CBP isolate from a subjectafflicted with carcinoma as an antigen can be used as an antigen. Mice,goats, rabbits, or other animals can be challenged by injection with asolution of such an isolate emulsified in complete Freund's adjuvant atweekly intervals. After the initial injection, the booster injectionscan be administered without adjuvant or emulsified in incompleteFreund's adjuvant. Alternatively, synthetic or genetically engineeredanalogs or fragments of the CBP produced by recombinant DNA orbiochemical techniques can be used as immunogens. An innoculumcontaining a relatively pure CBP sample isolated as described abovealong with Freund's adjuvent can be injected into a rabbit, mouse, rat,goat, or any mammal to produced monoclonal antibodies.

Monoclonal antibodies to a CBP or active binding fragments of suchantibodies can be generated by applying generally known cell fusiontechniques (cf. Kohler and Milstein (1976) Eur. J. Immunol. 6:511-519;and M. Shulman et al. (1978) Nature 276:269-270, herein incorporated byreference) to obtain a hybridoma producing the monoclonal antibody.Optionally, the monoclonal antibody may be subjected to proteolysis toobtain an active Fab, F(ab')₂, or Fv fragment.

More specifically, monoclonal antibodies are prepared by obtainingmammalian lymphocytes (preferably spleen cells), committing thelymphocytes to produce antibodies (e.g., by immunizing the mammal withthe particular antigenic determinant of interest beforehand), fusing thelymphocytes with myeloma (or other immortal) cells to form hybrid cells,and then culturing a selected hybrid cell colony in vivo or in vitro toyield antibodies which are identical in structure and specificity.

In particular, monoclonal antibodies to a CBP can be raised by employinga purified CBP isolate from a subject afflicted with carcinoma as anantigen. Mice or other animals can be challenged by injection with asolution of such whole cells emulsified in complete Freund's adjuvant atweekly intervals. After the initial injection, the booster injectionscan be administered without adjuvant or emulsified in incompleteFreund's adjuvant. Alternatively, synthetic or genetically engineeredanalogs or fragments of the CBP produced by recombinant DNA orbiochemical techniques can be used as immunogens.

Serum samples from the immunized animal can be analyzed by an enzymelinked immunoabsorbent assay ("ELISA") or the like for antibody reactionwith the immunization agent. Animals that exhibit antibodies titers aresacrificed and their spleens homogenized. Alternatively, the spleencells can be extracted and the antibody-secreting cells expanded invitro by culturing with a nutrient medium. The spleen cells are thenfused with myeloma (or other immortal) cells by the above-referencedprocedure of Kohler and Milstein. The hybridomas so produced arescreened (i.e., cloned by the limiting dilution procedure of theabove-referenced Baker et al. article) to select a cell line producingantibodies which react with human α chain receptor proteins.

Large scale antibody production can be obtained from suchanti-CBP-producing cell lines by various techniques, including theinduction of ascites tumors (e.g., after priming with pristane) and thepurification of such antibodies from the ascites fluid by ProteinA-Sepharose affinity chromotography. For a further description ofgeneral hybridoma production methods, see Oi and Herzenberg,"Immunoglobulin-Producing Hybrid Cell Lines" in Selected Methods inCellular Immunology (Mishell and Shiigi, Ed., W. H. Freeman & Co.,1980); and Scearce and Eisenbarth (1983) Meth. Enzymol. 103:459-469; andU.S. Pat. No. 4,411,933 issued to Gillis on Oct. 25, 1986, hereinincorporated by reference.

Human antibodies (i.e., those obtained from human-human or human-animalhybridoma) can be used as well as animal antibodies. For descriptions ofhuman hybridoma production techniques, see U.S. Pat. No. 4,451,570issued to Royston et al. on May 29, 1984; U.S. Pat. No. 4,529,694 issuedto Lazarus et al. on Jul. 16, 1985 and Zurawski et al., "ContinuouslyProliferating Human Cell Lines Synthesizing Antibody of PredeterminingSpecificity" in Monoclonal Antibodies (Plenum Press, New York 1980),also incorporated by reference.

Active antibody fragments can be derived from the monoclonal antibodiesdisclosed herein by a number of techniques. For example, purifiedmonoclonal antibodies can be cleaved with an enzyme such as pepsin, andthen subjected to HPLC gel filtration. The appropriate fractioncontaining Fab, F(ab)₂, or Fv can then be collected and concentrated bymembrane filtration or the like. For further description of generaltechniques for the isolation of active fragments, see for example, Khaw,et al., Vol. 23 J. Nucl. Med., pp. 1011-1019 (1982), incorporated byreference.

The antibodies and antibody fragments used herein can be labeledpreferably with radioactive labels by a variety of techniques other thanthe above-described Baker et al. technique. For example, thebiologically active molecules can also be labeled with a radionucleotidevia conjugation with the cyclic anhydride of diethylenetriaminepenta-acetic acid (DTPA) or bromoacetyl aminobenzyl ethylamine diaminetetra-acidic acid (BABE). See Hnatowich et al., Vol. 220 Science, pp.613-615 (1983) and Meares et al. (1984) Analytical Biochemistry. Vol.142, pp. 68-78, incorporated by reference for further description oflabeling techniques.

The instant invention also relates to a method for screening subjectsfor carcinoma. It includes subjecting a biological sample to at leastone test selected from a plurality of tests, each of which is specificfor a carcinoma cell. Useful biological samples include ascites fluid,whole blood, serum, bile, lymphoid tissue, or tumor tissue obtained froma subject afflicted with carcinoma. The method used to obtain thesesamples is dependent on the nature of the sample and would be known by amedical practitioner. Each test correlates the presence of a specificmarker with the presence of a carcinoma cell, and in some instances,with a degree of differentiation of that cell.

The screening method of the present invention includes tests for tumormarkers CEA, CA 19.9, NCA, AGP, CORA (U.S. patent application Ser. No.441,368, now U.S. Pat. No. 5,204,450), and the CC glycoprotein (U.S.Pat. No. 4,921,789), as well as any additional marker which indicate thepresence of carcinoma, such as CRP or SAP. The tests may be performed ina sequential manner until the presence of at least one marker has beenproven.

The tests performed may be assays, for example, to determineenzyme-linked activity, or may be immunoassays which utilize an antibodyspecific for a particular marker (see, e.g., U.S. Pat. No. 4,892,933).For example, an antibody raised to a cancer marker can be adhered to anadsorbent via chemical modification and/or covalent linkage using abifunctional cross-linking reagent.

This invention provides a convenient kit for screening biologicalsamples for colorectal carcinoma. This kit includes antisera or purifiedpolyclonal or monoclonal antibodies specific for the 20 kD and 21 kDCBPs, and antibodies for at least one other tumor marker such as the CCglycoprotein, CORA, NCA, CA 19.9, CEA, AGP, SAP, or CRP. Theseantibodies can be prepared by methods well known to those skilled in theart (see description above). Of course this kit may contain antigenbinding fragments of such antibodies such as Fv, Fab, or F(ab)₂fragments obtained by known proteolytic cleavage or recombinant DNAtechniques. Screening may be performed by any immunoassay proceduresknown in the art such as, for example, radioimmunoassay, Western blotanalysis, or nitrocellulose "dot" analysis.

The following examples illustrate the best mode of making and practicingthe present invention, but are not meant to limit the scope of theinvention, since alternative methods may be used to obtain similarresults.

EXAMPLE 1 Purification of CBPs

CBPs were isolated from ascites fluid from patients with colorectaladenocarcinoma using the following procedure. Exogenous CaCl₂ was addedto human ascites fluid to give a final concentration of 5 mM. Theascites was then incubated with an anti-CEA monoclonal antibody(Hybritech, San Diego, CA) coupled to an affinity gel (Affi-Gel 10;Bio-Rad). After an overnight incubation with agitation at 4° C., theimmunoadsorbent plus ascites fluid was put onto a (Affigel 10, Biorad,Richmond, Ca.) column, and any unbound material allowed to flow into awaste tube. The column was washed with wash buffer containing calcium(0.1 M Tris, pH 7.4, 0.15 M NaCl, and 5 mM CaCl₂) to remove anyunadsorbed molecules. CBP was eluted from the column using elutionbuffer containing EDTA (0.1 M Tris, pH 7.4, 0.15 M NaCl, 10 mM EDTA).

EXAMPLE 2 Analytical Methods

A. HPLC

CBP extracted from human ascites fluid as described above was passedthrough a size exclusion column (GF-250, DuPont). The eluates wereanalyzed with the mobile phase (0.2 M sodium phosphate, pH 7.0, 0.005%sodium azide) at a flow rate of 1 ml/min and detection at 280 nm. Theresulting elution profiles are shown in FIGS. 3A-3D. CBP migrates withCEA in the presence of calcium (FIG. 3C), while in the absence ofcalcium (presence of EDTA), it migrates as a separate peak (FIG. 3D).

B. Protein Sequencing

Samples were prepared for amino acid sequence analysis as follows. CBPspurified as described in EXAMPLE 1 were electrophoresed on anSDS-polyacrylamide gel (10-20% Mini-Gel, ISS, Newton, Ma.) according tothe method of Laemmli (Nature (1970) 227:680-685) The proteins on thegel were then electrotransferred to an Immobilon ™P Transfer Membrane(Millipore) with transfer buffer (25 mM Tris, pH 8.3, 192 mM glycine,and 15% methanol v/v) according to the method of Towbin et al. (Proc.Natl. Acad. Sci (USA) 76:4350-4354). After staining the membrane withCoomassie Blue, it was washed several times with distilled water toremove traces of transfer buffer. The photograph of the stained membraneis shown in FIG. 2. Two proteins are present having molecular weights of21 kD and 20 kD.

A duplicate gel was stained with periodic acid-Schiff base reagent (PAS)according to the method of Barber et al. (Biochem. (1971) 10:4711). Thisprocedure demonstrated that the 21 kD protein is glycosylated.

The CBP bands were excised from the gel and mechanically sequencedaccording to the method of Matsudaria (J. Biol. Chem. (1988,262:10035-10038) using an Applied Biosystems 477A protein sequencer. Theresulting sequence obtained from the 20 kD CBP is forth in the SequenceListing as SEQ ID NO: 4. The sequence obtained from the 21 kD CBP isshown in the Sequence Listing as SEQ ID NO:2.

EXAMPLE 3 Binding Assays

Affinity purified CBP was tested for the ability to bind to CEA in thepresence of calcium ions as follows. Varied amounts of CBP was mixedwith 5-10 μl CEA (100 μg/ml) CEA in the presence of 5 mM CaCl₂. Thesample was then run on an HPLC sizing column (DuPont GF-250), and theelution profile was recorded (FIG. 3C).

The appearance of a single peak with a retention time of about 8.5 minand the absence of a peak at about 9.4 min suggests the formation of acomplex between CEA and CBP. In control experiments (FIG. 3D) where CEAand CBP were mixed in the absence of Ca²⁺ (EDTA added), two peaks withretention times similar to that of CEA (FIG. 3A) and that of CBP (FIG.3B) were observed.

EXAMPLE 4 Isoelectric Focusing

The 20 kD CBP, the 21 kD CBP, CRP, and SAP were radioiodinated with ¹²⁵I using the chloramine T procedure of Greenwood et al (Biochem. J.(1963) 89:114-123) to procure a specific activity of about 6-10 mCi/mg.Focusing was carried out in agarose gels essentially as described bySaravis et al (Immunol Meth (1979) 29:91-96). Radiolabelled protein wasdetected by audoradiography using Kodak X-OMAT film.

EXAMPLE 5 Assay for CEA

NUNC-Immuno Plates (Naperville, IL) were coated with 500 ng/well CEA mAbin carbonate buffer (0.05 of sodium carbonate, ph 9.6), and wereincubated overnight (ON) at 4° C. The plates were washed with phosphatebuffered saline (PBS) containing 1% (wt/vol) bovine serum albumin (BSA)and 0.5% (vol/vol) Tween 20 (PBS-BSA-TWEEN). They were then treated with2% BSA in PBS for two hours at 4° C. to block any remaining sites on theplate. Antigen and high CEA serum standards were diluted and added tothe appropriate wells at 50 μl/well. The plates were incubated ON at 4°C., and then washed with PBS-BSA-TWEEN buffer. 50 μl/wellbiotin-labelled anti-CEA monoclonal antibody (lot no. E50713-103; 2.5μg/ml) was added and incubated for 3 hours at 37° C. The plates arewashed with PBS-BSA-TWEEN. Stepavidin peroxidase-conjugated horse radishperoxidase (HRP) was added (diluted according to the concentration ofthe lot), and the plate incubated for 1 hour at 37° C. The plates werethen washed with PBS-BSA-TWEEN. They were developed withorthophenylenediamine (Sigma, St. Louis, Mo.) and read with aspectrophotometer at 495 nm.

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The presentembodiments are therefore to be considered in all respects asillustrative and not restrictive, the scope of the invention beingindicated by the appended claims rather than by the foregoingdescription, and all changes which come within the meaning and range ofequivalency of the claims are therefore intended to be embraced therein.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 4                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 225 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (vii) FEATURE:                                                                (A) NAME/KEY: Serum amyloid P component                                       (x) PUBLICATION INFORMATION:                                                  (A) AUTHORS: Mantzouranis, Evanelia C.                                        Dowton, S. Bruce                                                              Whitehead, Alexander S.                                                       Edge, Michael D.                                                              Bruns, Gail A. P.                                                             Colten, Harvey R.                                                             (B) TITLE: Human Serum Amyloid P Component                                    (C) JOURNAL: Journal of Biological                                            Chemistry                                                                     (D) VOLUME: 260                                                                (E) ISSUE: 12                                                                (F) PAGES: 7752-56                                                            (G) DATE: 25 JUN 1985                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       MetGluLysLeuLeuLeuCysPheLeuValLeu                                             510                                                                           ThrSerLeuSerHisAlaPheGlyGlnThrAsp                                              1520                                                                         MetSerArgLysAlaPheValPheProLysGlu                                             2530                                                                          SerAspThrSerTyrValSerLeuLysAlaPro                                             3540                                                                          LeuThr LysProLeuLysAlaPheThrValCys                                            455055                                                                        LeuHisPheTyrThrGluLeuSerSerThrArg                                             6065                                                                          GlyTyrSerIlePheSerTyrAl aThrLysArg                                            7075                                                                          GlnAspAsnGluIleLeuIlePheTrpSerLys                                             8085                                                                          AspIleGlyTyrSerPheThrValGlyGlySer                                             90 95                                                                         GluIleLeuPheGluValProGluValThrVal                                             100105110                                                                     AlaProValHisIleCysThrSerTrpGluSer                                             115120                                                                        AlaSerGlyIle ValGluPheTrpValAspGly                                            125130                                                                        LysProArgValArgLysSerLeuLysLysGly                                             135140                                                                        TyrThrValGlyAlaGluAlaSerIleIleLeu                                             145 150                                                                       GlyGlnGluGlnAspSerPheGlyGlyAsnPhe                                             155160165                                                                     GluGlySerGlnSerLeuValGlyAspIleGly                                             170175                                                                         AsnValAsnMetTrpAspPheValLeuSerPro                                            180185                                                                        AspGluIleAsnThrIleTyrLeuGlyGlyPro                                             190195                                                                        PheSerProAsnValLeuAsnTrpArgAl aLeu                                            200205                                                                        LysTyrGluValGlnGlyGluValPheThrLys                                             210215220                                                                     ProGlnLeuTrpPro                                                               225                                                                           (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 40 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       XaaThrAspLeuSerGlyLysValPheValPhePro                                          510                                                                           XaaGluSerValThrAspXaaV alAsnLeuIleThr                                         1520                                                                          ProLeuGluLysProLeuGlnXaaPheThrXaaSer                                          253035                                                                        XaaXaaAlaTyr                                                                  40                                                                            (2) INFORMATION FOR SEQ ID NO:3:                                               (i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 223 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (vii) FEATURE:                                                                (A) NAME/KEY: C-reactive protein                                              (x) PUBLICATION INFORMATION:                                                  (A) AUTHORS: Lei, Ke-Jian                                                     Liu, Teresa                                                                   Zon, Gerald                                                                   Soravia, Emilia                                                                Liu, Teh- Yung                                                               Goldman, Neil D.                                                              (B) TITLE: Genomic Sequence for Human                                         C-reactive Protein                                                            (C) JOURNAL: Jouranl of Biological                                            Chemistry                                                                     (D) VOLUME: 260                                                               (E) ISSUE: 24                                                                 (F) PAGES: 13377-83                                                           (G) DATE: 25 OCT 1985                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       Met AsnLysProLeuLeuTrpIleSerValLeu                                            510                                                                           ThrSerLeuLeuGluAlaPheAlaHisThrAsp                                             1520                                                                          LeuSerGlyLysValPheValPheP roArgGlu                                            2530                                                                          SerValThrAspHisValAsnLeuIleThrPro                                             3540                                                                          LeuGluLysProLeuGlnAsnPheThrLeuCys                                             4550 55                                                                       PheArgAlaTyrSerAspLeuSerArgAlaTyr                                             6065                                                                          SerLeuPheSerTyrAsnThrGlnGlyArgAsp                                             7075                                                                          AsnGluLeuLeuVa lTyrLysGluArgValGly                                            8085                                                                          GluTyrSerLeuTyrIleGlyArgHisLysVal                                             9095                                                                          ThrSerLysValIleGluLysPheProAlaPro                                             100 105110                                                                    ValHisIleCysValSerTrpGluSerSerSer                                             115120                                                                        GlyIleAlaGluPheTrpIleAsnGlyThrPro                                             125130                                                                        Leu ValLysLysGlyLeuArgGlnGlyTyrPhe                                            135140                                                                        ValGluAlaGlnProLysIleValLeuGlnGly                                             145150                                                                        GluGlnAspSerTyrGlyGlyLysPheAspArg                                             155 160165                                                                    SerGlnSerPheValGlyGluIleGlyAspLeu                                             170175                                                                        TyrMetTrpAspSerValLeuProProGluAsn                                             180 185                                                                       IleLeuSerAlaTyrGlnGlyThrProLeuPro                                             190195                                                                        AlaAsnIleLeuAspTrpGlnAlaLeuAsnTyr                                             200205                                                                        GluIleArgGlyTyrValIleIleLy sProLeu                                            210215220                                                                     ValTrpVal                                                                     (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (vii) FEATURE:                                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       PheGly GlnThrLeuMetGlyGlyLysAlaPhe                                            510                                                                           ValPheProLysSer                                                               15                                                                        

We claim:
 1. An isolated protein that specifically bindscarcinoembryonic antigen (CEA), or a CEA-binding fragment of saidprotein,said CEA-binding protein (CBP) having a molecular weight ofabout 20 kD as determined by SDS-polyacrylamide gel electrophoresis, andbinding CEA in the presence of a divalent cation in vitro.
 2. Theprotein of claim 1 comprising the amino acid sequence set forth in theSequence Listing as SEQ ID NO:4.
 3. The protein or fragment of claim 1further comprising a label attached thereto.
 4. The protein or fragmentof claim 3 wherein said label is selected from the group consisting ofradioactive isotopes, enzymes, stable free radicals, coenzymes,fluorescent groups, chemiluminescent groups, toxins, and colorimetricgroups.
 5. An isolated protein that specifically binds carcinoembryonicantigen (CEA),said CEA-binding protein (CBP) having a molecular weightof about 20 kD as determined by SDS-polyacrylamide gel electrophoresis,and binding CEA in the presence of a divalent cation in vitro.
 6. Theprotein of claim 5 comprising the amino acid sequence set forth in theSequence Listing as SEQ ID NO:4.
 7. The protein of claim 5 furthercomprising a label attached thereto.
 8. The protein of claim 7 whereinsaid label is selected from the group consisting of radioactiveisotopes, enzymes, stable free radicals, coenzymes, fluorescent groups,chemiluminescent groups, toxins, and colorimetric groups.
 9. A devicecomprising:(a) a support; and (b) at least a CEA-binding fragment ofsaid CBP of claim 1 bound to said support.
 10. The device of claim 9wherein said CBP comprises the amino acid sequence set forth in theSequence Listing as SEQ ID NO:4.
 11. A method of detecting carcinomacomprising the steps of:(a) administering to a subject a pharmaceuticalformulation comprising a carcinoembryonic antigen binding polypeptide(CBP) or fragment thereof in a physiologically acceptable carrier, saidCBP having a molecular weight of approximately 20,000 daltons onSDS-polyacrylamide gels, and said CBP or said fragment thereof bindingCEA in the presence of a divalent cation in vitro; (b) obtaining abiological sample from said subject; (c) measuring the concentration ofCEA in said biological sample; and (d) comparing said CEA concentrationwith a predetermined threshold concentration of CEA indicative of thepresence of carcinoma.
 12. The method of claim 11 wherein saidadministering step (a) comprises administering to the circulation ofsaid subject a pharmaceutical formulation comprising a CEA-bindingfragment of said CBP.
 13. The method of claim 11 wherein saidadministering step comprises administering to the circulation of saidsubject a pharmaceutical formulation including a CBP comprising theamino acid sequence set forth in the Sequence Listing as SEQ ID NO:4.14. The method of claim 11 wherein said administering step comprisesadministering said formulation to said subject by injection.
 15. Themethod of claim 11 wherein said obtaining step comprises obtaining asample selected from the group consisting of whole blood, serum, bile,saliva, sputum, lymphoid tissue, tumor tissue, and ascites fluid. 16.The method of claim 11 wherein said measuring step further comprisesconducting an assay for CEA on said sample.
 17. The method of claim 16wherein said conducting step further comprises conducting an immunoassayfor CEA.
 18. An antibody or fragment thereof that specifically binds acarcinoembryonic antigen (CEA)-binding protein or CEA-binding fragmentthereof,said CEA-binding protein having a molecular weight of about 20kD as determined by SDS-polyacrylamide gel electrophoresis, and bindingCEA in the presence of a divalent cation in vitro.
 19. The antibody ofclaim 18 wherein said antibody is a monoclonal antibody.
 20. A method ofdetecting carcinoma comprising the steps of:(a) subjecting a biologicalsample from a subject to a test indicating the presence of a cancermarker, said marker comprising a carcinoembryonic antigen bindingprotein (CBP) that binds carcinoembryonic antigen (CEA) in the presenceof a divalent cation in vitro, and has a molecular weight of about20,000 daltons as determined by SDS-polyacrylamide gel electrophoresis,and (b) screening said sample for the presence of said CBP, the presenceof said CBP being indicative of the presence of carcinoma in saidsubject.
 21. The method of claim 20 wherein said subjecting step furthercomprises subjecting a biological sample to a test indicating thepresence of a cancer marker, said marker comprising the amino acidsequence set forth in the Sequence Listing as SEQ ID NO:4.
 22. Themethod of claim 20 wherein said subjecting step further comprises abiological sample to an immunoassay utilizing an antibody thatspecifically binds said CBP.
 23. The method of claim 20 wherein saidsubjecting step comprises subjecting a biological sample selected fromthe group consisting of whole blood, serum, bile, ascites fluid, saliva,sputum, lymphoid tissue, and tumor tissue to a test indicating thepresence of a cancer marker.
 24. A kit for screening a patient forcarcinoma, said kit comprising an antibody specific for acarcinoembryonic antigen binding protein (CBP) or a CBP-binding fragmentthereof, said CBP having a molecular weight of about 20,000 daltons onSDS-polyacrylamide gels, and binding carcinoembryonic antigen (CEA) inthe presence of a divalent cation in vitro.said kit further comprisingan antibody or binding fragment thereof specific for a carcinoma markerselected from the group consisting of carcinoma orosomucoid-relatedantigen (CORA), carcinoembryonic antigen (CEA), CA 19.9, non-specificcross-reacting antigen (NCA), and alpha-1 acid glycoprotein (AGP). 25.The kit a claim 24 wherein said antibody specific for said CBP is amonoclonal antibody.